Cholesterol is abundant in the retina, which maintains cholesterol homeostasis by balancing the pathways of cholesterol input and output. Retinal cholesterol input includes local biosynthesis and uptake from the systemic circulation. Retinal cholesterol output is realized via photoreceptor phagocytosis, metabolism to oxysterols by cytochrome P450 enzymes, and transport to the systemic circulation by lipoproteins. Elaborate mechanisms control and coordinate retinal cholesterol input and output to maintain lipid steady-state levels. Accumulating data implicate retinal cholesterol dyshomeostasis in the pathogenesis of age-related macular degeneration (AMD), the leading cause of vision loss in the elderly of the industrialized world. The details of the cholesterol- AMD link are, however, still unclear, due to insufficient knowledge about retinal cholesterol maintenance. During the previous grant period, we ascertained: 1) the relative contributions of retinal cholesterol biosynthesis and uptake of systemic cholesterol to the total retinal cholesterol input; 2) retinal significance of cholesterol transport and storage; and 3) the effect of different pharmacologic treatments on lowering retinal cholesterol. Specifically, we found that local biosynthesis is the major source of retinal cholesterol in mice, and a pathway, which can be inhibited by a cholesterol lowering drug simvastatin. Furthermore, we established that apolipoproteins E and D are important for retinal cholesterol transport, a pathway which could be targeted pharmacologically as well and lead to retinal cholesterol lowering. Finally, we discovered that cholesterol excess could be esterified in the retina and form lipid droplets, i.e., identified a mechanism for managing retinal cholesterol overload. In this renewal we will continue to delineate the unknown aspects of retinal cholesterol maintenance that are of unquestionable importance for our understanding of how to combat AMD. Aim 1 will evaluate hamsters as a model for studies of retinal cholesterol. Among rodents, hamsters are much closer to humans than mice in terms of their whole body cholesterol maintenance. Hence, we will investigate whether there is any advantage in using hamsters for establishing the details of retinal cholesterol homeostasis as well as pharmacologic treatments. Aims 2 and 3 will still use mice as studies under these Aims should not be affected by potential interspecies differences in retinal cholesterol maintenance. Aim 2 will focus on 2-hydroxypropyl-b-cyclodextrin, the FDA-approved pharmaceutical that targets cholesterol distribution. The pharmacologic potential of this cholesterol-related process for retinal cholesterol lowering has not yet been investigated and will be tested on several mouse models. Aim 3 will establish retinal significance of apolipoprotein J for the pathway of retinal cholesterol transport. Several apolipoproteins appear to be necessary for cholesterol trafficking in the retina, including apolipoprotein J, a protein with unique functions. Collectively, the three Aims will provide principally new information about cholesterol in the retina and facilitate the development of new therapeutics for the diseases associated with deleterious accumulations of cholesterol in the retina and Bruch?s membrane.